Trolox

The antioxidant activity was evaluated in each JF sample by the Trolox Equivalent Antioxidant Capacity (TEAC) method, adapted for the Infinite 200 PRO microplate reader (Tecan, Mannedorf, Switzerland) using the radical cation ABTS•+ and Trolox (Sigma-Aldrich, Darmstadt, Germany) as standard (49 (link), 50 (link)). The samples and the standard were assayed under the same conditions as already described in De Domenico et al. (40 (link)). The results were expressed as nmol of Trolox Equivalents per gram of fresh weight (nmol TE/g FW), and means of at least three measurements from two independent experiments were considered.

For ORAC the method of Davalos et al. [82 (link)] was used. Extracts were diluted with 75 mM phosphate buffer (pH 7.4). The assay was carried out in black-walled 96-well plates (Greiner-Bio One) and each well contained a final volume of 200 μL. To each well 20 μL of extract and 120 μL of fluorescein (FL; 70 nM final concentration) were added and the plate was incubated at 37 °C for 15 min. The AAPH (60 μL; 12 mM final concentration) was added to each well and fluorescence intensity was estimated using an Infinite200 Pro plate reader (Tecan, Männedorf, Switzerland), every minute for a total of 80 min using an excitation wavelength of 485/9 nm and an emission wavelength of 535/20 nm. A standard curve was constructed using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-Aldrich, Oakville, ON, Canada, 1.5–10.5 μM). A blank (fluorescein + AAPH) using phosphate buffer instead of the antioxidant solution was carried out in each assay. Results were determined by using Magellan v 7.2 software (Tecan, Männedorf, Switzerland), on the basis of the difference in area under the curve between the control and the sample and expressed as μmoles of Trolox equivalents (TE) per g of lipidic extract. All the reaction mixtures were prepared in triplicate and at least three independent assays were performed for each sample.

Anti-oxidative status was measured as described before [35 (link),36 (link)]. Shortly, a 7 mM ABTS radical stock solution was prepared by diluting 19 mg of 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma A1888) (ABTS+.) and 20 mg ammonium persulfate (Sigma A3678) in 5 mL distilled water at room temperature. The stock solution was further diluted 1:4 in distilled water to obtain an ABTS working solution. As standard either Trolox (Sigma 238813, Austria, St. Louis, MO, USA), quercetin Q2 or FeQ2 was used. Samples of apoBet v 1 (0.5 mg/mL) and/or preincubated with FeQ2, Q2 or ferroxamine (FO) were subjected to gel filtration (PD MiniTrap G-25 Columns, GE Healthcare 28-9180-07) to remove unbound ligands. 50 µL of diluted samples or diluted standards (Trolox, Q2, FeQ2) were incubated with 50 µL of ABTS working solution for 2 min in a 96 well plate before measurement of absorbance at 740 nm using an Infinite M200Pro microplate reader (Tecan, Grödig, Austria). Data are shown as antioxidative capacity using Trolox-equivalent normalized to apoBet v 1 in Figure 1f,g using FeQ2 and Q2 as standard to calculate the molar ratio of quercetin to Bet v 1.